Despite advances in our understanding of the TCR/MHC interaction, the biophysical basis of the increased sensitivity of activated T cells was poorly understood. Our recent studies have started to provide a biophysical basis for this enhanced sensitivity. Using peptide loaded MHC-Ig (pepMHC-Ig) dimers, we found that upon activation, T cells develop increased avidity for antigen. This change depends on increased TCR crosslinking and is sensitive to the cholesterol content of the T cell membrane. Activation-induced membrane changes in TCR avidity represent a previously unrecognized means of increasing the sensitivity of activated T cells to antigen. The current proposal will define, mechanistically, the biophysical changes that result in increased TCR avidity, its control by cholesterol, and the physiological importance of TCR crosslinking. Mechanistically, we will determine if activation induced TCR membrane reorganization leads to preformed TCR dimers/oligomers or results from increased crosslinking potential due to changes in membrane fluidity. Further, we hypothesize that CD8 plays a central role in the enhanced MHC-Ig binding seen with T cell activation due to a change in the relationship of CD8 to the TCR complex. Our preliminary data show that changes in cell membrane cholesterol alter both MHC-Ig binding as well as TCR-mediated calcium signaling. To further analyze the effect of cholesterol we will determine the mechanism of action of cholesterol, the link between cholesterol addition and cytoskeleton, and the effects of cholesterol on other aspects of activation, such as changes in cell surface markers, raft formation, and cytokine expression. The physiologic significance of activation induced TCR membrane reorganization will be further defined by analyzing enhanced TCR avidity in in vivo stimulated acutely activated T cells, memory T cells, and "dysfunctional" T cells. We hypothesize that only when stimulation leads to development of effective immune responses will T cells show enhanced TCR avidity. These studies will further our understanding of increased TCR avidity as a general mechanism for facilitating immune recognition of antigen. [unreadable] [unreadable]